KR20170107459A - A ball-shaped matrix as a cell carrier - Google Patents

Abstract

The present invention provides a method for preparing a pharmaceutical composition comprising the steps of: (a) providing an initial composition comprising a cross-linked fibrinogen and a fibrin-free, ball-shaped matrix; and (C) mixing the emulsion at a temperature of from 50 캜 to 80 캜 until a ball-shaped matrix is obtained, and (d) mixing the resultant matrix ≪ RTI ID = 0.0 > a < / RTI > The matrix is used as a cell carrier.

Description

A ball-shaped matrix as a cell carrier

The present invention relates to a ball-shaped matrix, a method for producing such a matrix and its use as a cell carrier.

The present invention is applied in the field of cell culture, and in particular in the field of cell proliferation used in the context of cell therapy, biogenesis to produce biologically active molecules, or investigation and development as a cell model. Such cells are, for example, primary cells, pluripotent stem cells, stem cell lines or inactivated cell lines.

In particular, cell therapy based on the use of adult stem cells may be selected and / or administered by administering healthy and functional cells that are amplified and / or modified in vitro Replacement and regeneration of cells in unhealthy tissues.

Since different stem cell sources do not allow a sufficient amount of cells for clinical use, a cell proliferation step is necessary.

Currently, adherent stem cells are cultured in two-dimensional systems, especially plastic culture vessels.

By using a large number of culture vessels, for example, by increasing the size of the culture vessel, or by using a multilevel culture vessel such as Celltack (trade name) (Corning (USA)) to obtain a larger volume of cells, It is necessary to use a culture surface. In this case, however, handling of the culture vessel is more laborious.

The use of a three-dimensional culture system, particularly a microcarrier in suspension, is also tested.

For example, document US 2012/0009645 suggests a method of culturing pluripotent stem cells (embryonic stem cells, inducible pluripotent stem cells) on microbeads in the presence of ROCK inhibitors. Microbeads are, for example, D53 cellulosic beads or Cytodex 1 or 3 beads.

However, the use of such synthetic microcarriers has several disadvantages. Above all, the microcarriers have a low rate of cellular inoculation (less than 30%), resulting in a massive loss of cells of interest from the onset of the proliferative process. Next, cell desorption and carrier-cell separation from its carrier prior to injection into the patient remains a significant problem. Finally, when this synthetic microcarrier is used, it can not be blocked and thus emits plastic particles that can be injected into the patient simultaneously with the cells of interest.

To counteract the problem of cell attachment, it has been proposed to coat microcarriers by molecules. However, this molecule is usually expensive / expensive or animal origin, which poses a biological safety problem.

For example, document US 2011/0111498 proposes a method of culturing stem cells (mesenchymal stem cells, embryonic stem cells or inducible pluripotent stem cells) on a microcarrier in suspension. Microcarriers are, for example, synthetic beads (Toyopearl, Cytodex, DE52) with a coating of the Matrigel (TM), laminin or hyaluronic acid type capable of supporting positive charge and cell growth.

Document WO 2009/134197 also describes microcarriers for cell culture, such as Cytodex, coated with plant protein hydrolysates such as soy peptone hydrolyzate. The microcarriers have the advantage of not containing animal proteins.

Document WO 2011/017050 has an animal origin protein for cell culture with a polymer coating (e.g., swollen methacrylate) grafted with a polypeptide comprising an RGD sequence that facilitates cell attachment Do not offer microcarriers.

There are also cell microcarriers based on natural ingredients.

For example, document EP 1,015,570 from International Patent Application WO 99/15637 proposes fibrin (fibrinogen) microbeads as cell carriers. This microbead has a high degree of cross-linking of the fibrin (fibrinogen) obtained during the propagation of the microbeads from fibrinogen, thrombin and factor XIII. However, thrombin used as a cross-linker is exogenous, which increases manufacturing difficulties and biological risks.

In Journal of Biotechnology, 2010, vol. 146, no. 4, pp. 194-197), gelatin microspheres were prepared by the method described in Eibes G. et al. (Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier- It has been found that porous beads (Cultispher S) can attach mesenchymal stem cells and make them proliferate.

Lastly, Oliveira MB et al. (Injectable PEGylated fibrinogen cell-laden microparticles made with a continuous solvent- and oil-free preparation method. Acta Biomaterialia, vol. 13, Nov. 18, 2014, pp. 78-87) Discloses pegylated fibrinogen microparticles. The microparticles obtained by UV irradiation and by running the continuous manufacturing process in the absence of solvent and oil allow 3D cell culture and provide a platform for encapsulation of cells with high water content.

The present invention proposes a three-dimensional biocompatibility matrix for cells that results in an adhesion rate of greater than 75%. This matrix is further easily degradable so that cells can be recovered. The matrix also has the advantage that it can be produced from a single biological product without requiring a protein or other exogenous material.

To this end, according to a first aspect, the invention relates to a ball-shaped matrix comprising crosslinked fibrinogen, said matrix not containing fibrin.

According to a second aspect, the present invention relates to a method of manufacturing a matrix according to the first aspect,

(a) providing an initial composition comprising fibrinogen and a platelet factor;

(b) injecting the initial composition into a heated oil at a temperature of from 50 캜 to 80 캜 to form an emulsion,

(c) mixing said emulsion at a temperature of from 50 캜 to 80 캜 until a ball-shaped matrix is obtained, and

(d) separating the thus obtained matrix.

According to a third aspect, the invention relates to the use of a matrix according to the first aspect as a cell carrier.

Other objects and advantages will be apparent through the process of the following description.
FIGS. 1 and 2 show photographs of a porous matrix according to the present invention, respectively, using scanning electron microscopy.
Figure 3 shows photographs of the matrix according to the invention in an incubation medium and in the presence of cells by optical microscopy (20 magnification).

According to a first aspect, the present invention relates to a ball-shaped matrix comprising crosslinked fibrinogen. The matrix is particularly free of fibrin.

"Matrix" means a solid or semi-solid three-dimensional structure that a cell can attach and / or proliferate.

The matrix of the present invention is in the form of a ball, i.e. it is spherical or spheroidal. It may have a regular or irregular shape.

The matrix does not contain fibrin and includes crosslinked fibrinogen, which allows obtaining a mechanically stable matrix that is not degraded during cell culture.

Fibrinogen is a soluble protein present in plasma. The fibrinogen protein consists of three pairs of homologous peptide chains linked by a disulfide bridge.

Crosslinked fibrinogen is a fibrinogen to which at least two sites of the polypeptide chain are connected. Crosslinking is the intermolecular (the linked site then belongs to the same fibrinogen) and / or the extramolecular molecule (the two or more fibrinogen molecules are then linked). Crosslinked fibrinogen is insoluble in water and organic solvents.

During in vivo, plasma clotting, fibrinogen is degraded by the action of thrombin to fibrin monomers. The fibrin monomer will then polymerize into fibrin, which is unstable. The factor XIIIA will finally allow its cross-linking to stabilize the fibrin polymer.

The cross-linking of fibrinogen is consequently an unnatural process, surprisingly obtained by the applicant, from a particular initial composition. The crosslinked fibrinogen matrix of the present invention is a synthetic fibrinogen matrix, which does not contain fibrin, especially with a non-natural three-dimensional structure in the form of a ball.

Advantageously, the fibrinogen contained in the matrix is not physically and / or chemically functionalized, for example by pegylation. The fibrinogen contained in the initial composition is natural fibrinogen that is not modified by humans.

According to one particular embodiment, the initial composition comprises soluble fibrinogen. More particularly, the initial composition comprises plasma, which is a fibrinogen source. Even more particularly, the plasma is human plasma.

In particular, fibrinogen is cross-linked by platelet factors.

Platelet factor refers to one or several species of substances contained or secreted by platelets. In particular, the platelet factor is a substance contained in platelet lysate.

Platelet lysate refers to the product of the dissolution of platelets, i. E., The product obtained after the disruption of the cell membrane, which results in the release of molecules normally contained in platelets (growth factors, cytokines).

Dissolution of the platelets is obtained, for example, by one or several cycles of refrigeration / thawing, ultrasonic or solvent / detergent treatment.

Crosslinking of fibrinogen is made possible by the presence of platelet factors. The following described method of preparation performed from the initial plasma composition without platelets in place of the initial platelet lysate composition did not result in matrix formation. Without exogenous thrombin, and thus without fibrin formation, cross-linking of the fibrinogen is due to the presence of one or several platelet lysates.

In particular, cross-linking takes place without the need to add chemicals, such as glutaraldehyde or carbodiimide derivatives or exogenous substances such as non-human thrombin, genipin or sugar.

The matrix thus has the advantage of containing only proteins of human origin. The matrix is therefore biocompatible, i.e. non-toxic with respect to cells, tissues and organs.

According to one embodiment, the matrix has a diameter comprised between 20 μm and 2 mm, in particular between 50 μm and 1000 μm. For example, the matrix has a diameter comprised between 200 μm and 800 μm.

In particular, the matrix according to the invention is a porous matrix, i. E. It has a plurality of gaps or orifices in its structure.

In particular, the matrix according to the present invention has pores having a diameter of less than 1 탆.

The pore size of the matrix is verified using electron microscopy techniques.

When the matrix of the present invention is fibrinogen-based, it has cell attachment properties.

Due to the small size of the pores of the matrix, the cells attached to it remain on its surface and do not migrate inside. This porous matrix can also adsorb small bioactive substances such as growth factors, cytokines, hormones or other active ingredients.

According to a particular embodiment, the matrix further comprises at least one biologically active substance such as a cell, a protein, for example a growth factor, a medical product, a hormone, a cytokine or a combination thereof.

The biologically active material is encapsulated, absorbed, adsorbed and / or covalently bound to the matrix.

In a particular example, the matrix comprises primary cells and / or cell lines. Examples of such cells are stem cells, fibroblasts, endothelial cells, cartilage cells, neuroblastoma cells, kidney cells, liver cells, pancreatic cells, thyroid cells, glial cells, muscle cells, mouse breast carcinoma cells and combinations thereof. Even more particularly, the cells are CHO (Chinese hamster ovary) cells, Vero cells or mesenchymal stem cells.

In another specific example, the matrix does not comprise human embryonic stem cells.

According to one particular embodiment, the cell is infected with a virus, a gene expressing the recombinant protein of interest, or an exogenous DNA.

In particular, the matrix further comprises at least one growth factor, such as IGF-1 (insulin growth factor-1). More particularly, the growth factor is endogenous, i. E. It is included in an initial composition comprising fibrinogen intended to form a matrix.

The concentration of endogenous growth factor (s) in the matrix varies as a function of the manufacturing method and / or the initial concentration of growth factor (s) in the initial composition.

The matrix according to the invention has the advantage that it can be completely degraded by enzymatic and / or chemical action. This property facilitates the recovery of cells after their proliferation.

For example, enzyme or non-enzymatic cytolytic substances such as trypsin, trypsin-EDTA combination, Accutase TM (Chemicon) or TrypLE TM (Gibco) are used to degrade the matrix and thus detach cells therefrom.

According to yet another aspect, the present invention relates to a method of manufacturing a matrix according to the first aspect,

(a) providing an initial composition comprising fibrinogen and a platelet factor;

(b) injecting the initial composition into a heated oil at a temperature of from 50 캜 to 80 캜 to form an emulsion,

(c) mixing said emulsion at a temperature of from 50 캜 to 80 캜 until a ball-shaped matrix is obtained, and

(d) separating the thus obtained matrix.

The starting product of the method is an initial composition comprising fibrinogen and a platelet factor. In particular, the initial composition comprises plasma as a fibrinogen source and platelet lysate as a platelet factor source. Moreover, the initial composition does not contain exogenous thrombin, which simplifies manufacturing and reduces biological risks.

Advantageously, the plasma source and the platelet lysate source are shared. In this case, the initial composition is platelet lysate obtained by dissolution of platelets in the suspension in plasma. For example, the initial composition is a platelet lysate obtained by dissolving one or several platelet concentrates or one or several platelet-rich plasma.

According to one alternative, the platelet concentrate comprises a platelet preservative additive solution. In this case, the amount of plasma in the platelet concentrate, and hence the amount of fibrinogen, must remain sufficient to permit matrix formation.

In order to support the spherical system and reduce the aggregation of the matrix, the initial composition additionally comprises a surfactant.

"Surfactant" refers to an amphipathic molecule, one having two moieties with lipophilic (miscible in oil) and nonpolar and the other having a different polarity that is hydrophilic (miscible with water) and polar.

Surfactants act to facilitate the formation of emulsions, stabilize them, and promote dispersion of the formed droplets.

Surfactants are anionic, cationic, amphoteric or nonionic. Examples of surfactants are, in particular, sodium dodecyl sulfate, Triton X-100, Tween 20, Tween 80 and combinations thereof.

In particular, the composition comprises from 0.5% to 20%, in particular from 1% to 5%, of a surfactant.

The method then involves the injection of the initial composition into an oil heated to a temperature comprised between 50 캜 and 80 캜 to form an emulsion. A small droplet will then be formed and dispersed in the hot oil.

According to one particular embodiment, the oil is an animal or vegetable oil, in particular a plant oil, such as sunflower, olive or canola oil.

Under the influence of heat, it is presumed that the inaccessible binding site of the fibrinogen protein is exposed and allows crosslinking so that fibrinogen is insoluble in water and organic solvents.

If the initial composition is a platelet lysate obtained by dissolution of platelets in suspension in plasma, the cross-linking of the fibrinogen is produced essentially, i.e. without the use of external substances.

The thus obtained emulsion is mixed next to form a matrix.

The way the emulsion mixes affects the matrix. In particular, the mixing of the emulsions is carried out by stirring at a rate comprised between 100 and 1500 cycles per minute, especially over 700 cycles per minute. The larger the agitation speed, the smaller the matrix will be.

The mixing is carried out for a time comprised between 2 hours and 10 hours, in particular between 3 hours and 5 hours.

By varying the amount and type of surfactant and / or by varying the agitation rate, the size of the matrix can be controlled.

The step of isolating the formed matrix is carried out by filtration followed by continuous washing of the matrix with an organic solvent to remove trace amounts of oil.

The method forms a matrix of different sizes. Alternatively, the method comprises a step of filtering the matrix through a sieve, followed by isolation of the matrix, accompanied by a reduction in size, to obtain a corrected matrix.

The process for obtaining the matrix according to the invention is a non-natural process which requires a contribution heat of over 50 [deg.] C.

The matrix obtained by this method from platelet lysates is porous, consisting essentially of cross-linked fibrinogen, and free of fibrin.

According to a third aspect, the invention relates to the use of a matrix according to the first aspect as a cell carrier.

For cell culture, the matrix is placed in a suspension in a culture medium contained in a culture vessel. The cells to be cultured are then inoculated, i. E. Placed in contact with the matrix.

For example, the matrix is used for cell proliferation, production of proteins of interest, and cell regeneration.

The matrix is biocompatible, so it can be used with or without the biologically active material in vivo.

One particular advantage of the matrix according to the invention is its very high cell attachment power. In fact, more than 75% of the cells adhere to the matrix during the inoculation step.

Other fields for the matrix according to the present invention are, for example, tissue regeneration, scarring, controlled release of medical products or stem cell selection.

Example

Manufacture of balls

200 ml of sunflower oil was heated in a 500 ml vessel under magnetic stirring until reaching 65 캜. 50 ml of platelet lysate was then added to the warm oil. The thus obtained solution was kept under stirring at 65 DEG C until the balls appeared. Heating of the formulation was then stopped and stirring was maintained until it returned to ambient temperature. The formulation was then filtered in a sieve with a calibrated size and the recovered balls were then washed three times with organic solvents (xylene, hexane and ethanol) and placed in a saline solution.

Due to its particular structure, the ball can also be maintained in its powdered form. The balls may then be stored at ambient temperature or at 4 ° C for long term storage.

The ball thus obtained is spherical and has a diameter of 30 占 퐉 to 2000 占 퐉. By screening, a population of balls having a desired size can be obtained.

Scanning electron microscopy analysis showed dense structures allowing cell attachment (FIGS. 1 and 2).

Inoculation of the ball with cells

The balls in solution were placed in contact with a known number of mesenchymal stem cells in a small volume of complete culture medium (10 ml). Cells attach to the ball (Figure 3).

After 4 hours of contact in a thermostat at 37 占 폚, the (ball-free) culture medium was collected and centrifuged. The number of cells that did not adhere to the ball found in the supernatant was counted, allowing a cell attachment rate of 75% to be determined.

Decomposition of balls for cell recovery

The balls filled by the cells were kept in culture to obtain sufficient cell numbers. At the end of the cell culture period, the ball / cell complex was recovered by centrifugation of the whole culture medium. The recovered pellet, consisting of the ball / cell complex, was washed twice in PBS. After the washing operation, an enzyme (TrypLE TM), which allows complete degradation of the balls without causing cell death, was added to the pellet and placed at 37 ° C for 20 minutes. After complete digestion of the balls, the solution can be centrifuged and the recovered cells can be used subsequently.

Cell proliferation rate

The cell proliferation test in the balls according to the present invention was performed in comparison with polystyrene balls.

Both cell lines and human primary cells were inoculated with the same density on both types of balls. The amount of unattached cells was assessed to accurately determine the amount of attached cells.

Cells on the balls were kept in culture for 10 days by the addition of fresh medium every 3 days. At the end of incubation, the ball / cell complex was recovered and the total amount of cells generated was assessed.

The test was designed to show cell multiplication of 23-fold (versus 30-fold versus polystyrene ball) and 14-fold (versus 12-fold versus polystyrene ball) versus human primary cell line Were allowed to the present inventors.

Identification of balls

After manufacturing the ball, it was disassembled to study its composition precisely. Immunological assays allowed the balls according to the invention to be predominantly composed of fibrinogen (on average, present at 100 占 퐂 / ml). The assay allowed the presence of a concentration of 0.3 pg / ml of IGF-1 in the balls according to the present invention.

The presence of fibrin was not detected in the ball.

Claims (17)

A ball-shaped matrix comprising crosslinked fibrinogen,
≪ / RTI > wherein the matrix is free of fibrin. The matrix of claim 1, wherein the matrix is porous. 3. The matrix of claim 2, having pores having a diameter of less than 1 mu m. 4. The matrix according to any one of claims 1 to 3, characterized in that the fibrinogen is crosslinked via platelet factor. 5. Matrix according to any one of claims 1 to 4, characterized in that it has a diameter comprised between 20 μm and 2 mm, in particular between 50 μm and 1000 μm. 6. The method according to any one of claims 1 to 5, further comprising at least one biologically active substance such as a cell, a protein, for example a growth factor, a medical product, a hormone, a cytokine or a combination thereof As a matrix. 7. The matrix of claim 6, wherein said protein is a growth factor, such as IGF-1. 8. The matrix of any one of claims 1 to 7, wherein cells are attached to the surface of the matrix. 9. A method of manufacturing a matrix according to any one of claims 1 to 8,
(a) providing an initial composition comprising fibrinogen and a platelet factor;
(b) injecting the initial composition into a heated oil at a temperature of from 50 캜 to 80 캜 to form an emulsion,
(c) mixing said emulsion at a temperature of from 50 캜 to 80 캜 until a ball-shaped matrix is obtained, and
(d) separating the resulting matrix. 10. The method of claim 9, wherein the initial composition is a platelet lysate obtained by dissolving platelets in suspension in plasma. 11. The method of claim 10, wherein the platelet lysate is obtained from one or several platelet concentrates. 12. A method according to any one of claims 9 to 11, characterized in that the initial composition further comprises a surfactant. 13. A process as claimed in claim 12, characterized in that the composition comprises from 0.5% to 20%, in particular from 1% to 5%, of a surfactant. 14. The process according to any one of claims 9 to 13, characterized in that the mixing of the emulsion is carried out by stirring at a rate comprised between 100 and 1500 cycles per minute. 15. The process according to any one of claims 9 to 14, characterized in that the oil is a vegetable oil, in particular an olive oil. 16. A process according to any one of claims 9 to 15, characterized in that the mixing of the emulsion is carried out for a period of time comprised between 2 hours and 10 hours. Use of a matrix according to any one of claims 1 to 8 as a cell carrier.

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